The long term objectives of this proposal are to determine of the mechanism by which the HIV envelope glycoprotein (Env) catalyzes the fusion of biological membranes. In this study we will focus on the role that the membrane-spanning (TM) domain of the gp41 subunit of the envelope protein plays in the fusion process. The specific aims are: (1) To determine the N-terminal boundary of the membrane-spanning domain of the HIV envelope protein. (2) to determine the structural features in the membrane-spanning domain of the HIV Env protein required for membrane fusion activity, and (3) To determine whether mutations that affect cell fusion activity also have an effect on virus penetration and infectivity. Given the critical roles in virus replication, the HlV Env glycoproteins are suitable targets for antiviral drug development and therapy; thus, compounds or strategies that specifically interfere with the virus-induced membrane fusion process offer therapeutic potential. However, progress in this area of antiviral research has been slow, primarily because the precise mechanism by which viral fusion proteins function has not been determined. The HIV Env proteins have uncommon TM domains, yet little is known about their structural and functional significance. Because the boundaries of the TM domain have not yet been adequately mapped, the first series of experiments will be to determine the N-terminal boundary directly by protease digestion of the ectodomain followed by amino acid sequencing of the residues protected by the membrane environment. Molecular modeling reveals that the HIV-1 TM domain has the potential to form an amphipathic alpha-helix. We will test the hypothesis that an amphipathic TM domain is required for the fusion function of Env using site-directed mutagenesis. Finally, each of the mutant envelope proteins that have already been generated in the preliminary studies, and those that are proposed, will be analyzed for their ability to support virus infection using a replication-defective HIV complementation assay, and a replication competent HIV system.